A Aggregation inhibitors. Part 1: Synthesis and biological
activity of phenylazo benzenesulfonamides
Shwu-Jiuan Lin,
aYoung-Ji Shiao,
cChih-Wen Chi
dand Li-Ming Yang
a,b,*
aDepartment of Medicinal Chemistry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing St., Taipei 110, Taiwan bDivision of Medicinal Chemistry, National Research Institute of Chinese Medicine, 155-1 Li-Nong St., Sec. 2,
Taipei 112, Taiwan
cDivision of Pharmacology, National Research Institute of Chinese Medicine, 155-1 Li-Nong St., Sec. 2,
Taipei 112, Taiwan
dInstitute of Pharmacology, National Yang-Ming University, 155 Li-Nong St., Sec. 2, Taipei 112, Taiwan
Received 18 November 2003; accepted 16 December 2003
Abstract—Phenylazo benzenesulfonamides were designed and synthesized as b-amyloid (Ab40) fibril assembly inhibitors, and
eval-uated for inhibition of Ab40aggregation and neurotoxicity using rat cortical neurons. Compound 2 (LB-152) was the most potent
compound in this study, and the para-NMe2group on the end of the phenylazo moiety may play an important role in preventing Ab40
fibril formation. LB-152 provides a new lead for further development of potential b-amyloid aggregation inhibitors to treat AD. #2004 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) neuropathology is characterized by extracellular amyloid deposition of senile plaques and neurofibrillary tangles in vulnerable AD brain regions.1 Clinical symptoms of AD include cognitive decline, irreversible memory loss, disorientation, language impairment, etc. Because this devastating illness affects a large number of older patients and their families and no effective treatment or cure currently exists, better diagnosis and treatment of AD are urgently needed. Although the mechanism by which amyloid fibril form-ation is associated with AD dementia is not completely understood, recent research suggests that aggregation of the amyloid-b peptide (Ab) into macromolecular b-sheet fibrils plays a causal role in the pathogenesis of AD.2 Suppression of this transition from monomeric to poly-meric Ab aggregation is believed to be a singular therapy for AD.3Until now, much research has focused on the discovery and development of non-peptide small-molecule Ab aggregation inhibitors for clinical evaluation in AD treatment.4,5
Small-molecule inhibitors from various chemical classes have recently been reported and reviewed.6,7 These
compounds inhibit or reduce in vitro aggregation of Ab and are somewhat similar in structure to biphenyl naphthyl diazo dyes such as Congo red (CR) and related sulfonate anions, which reportedly disrupt Ab aggregation and reduce Ab toxicity.8,9Unfortunately, the therapeutic potential of CR is severely diminished by the fact that it does not cross the blood–brain barrier (BBB).10Also, a naphthyl monoazo derivative1(Fig. 1), a small-molecule probe for inhibition of Ab40 aggregation, has been extensively discussed.11 These facts suggest that naphthylsulfonamide azo dyes can provide an appropriate starting point for future efforts to develop inhibitors of Ab aggregation.12
In the process of seeking new leads for Ab40aggregation inhibitors, we employed the concept of bioisosterism to design phenylazo benzenesulfonamide derivatives (Fig. 2). To our knowledge, no phenylazo benzenesulfonamide derivative has been reported to inhibit Ab40aggregation; however, we previously reported the synthesis of a series of trans-stilbene benzenesulfonamide derivatives as potent antitumor agents.13 Based on the principle of isosteric replacement,14 the C,C double bond in the trans-stilbene benzenesulfonamides can be interchanged with the N,N double bond in phenylazo benzenesulfon-amide derivatives. Consequently, the structure of the reference inhibitor 1 was also simplified by replacing the naphthylazo moiety with a phenylazo group. Thus, 0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.086
Bioorganic & Medicinal Chemistry Letters 14 (2004) 1173–1176
Keywords: beta-Amyloid; Alzheimer’s disease; Phenylazo; Benzene-sulfonamide.
* Corresponding author. Tel.: 2820-1999x8551; fax: +886-2-2826-4276; e-mail: lmyang@nricm.edu.tw
new phenylazo benzenesulfonamides were synthesized and evaluated for Ab40 aggregation inhibitory activity and neurotoxicity.
The synthesis of the target compounds (1–5) is outlined inSchemes 1–3. Although the synthesis of these deriva-tives has already been reported,15only a limited series of examples has been described, and neither the biological activity nor the possible chemical conversion was inves-tigated. This process involved initial diazotization of sulfanilamide with sodium nitrite in hydrochloride solution to afford the diazonium salt 6, followed by coupling with the molar equivalent of N,N-dimethyl-aniline or 3-(methylthio)N,N-dimethyl-aniline, in the presence of sodium acetate solution at 0C. The reactions were
pre-ferentially formed with a change in color from pale yellow to deep brown or reddish orange to afford the corres-ponding phenylazo benzenesulfonamide derivatives 2 and 3 (Scheme 1).
However, attempts to use this general approach for the coupling of diazonium salt 6 with aniline to produce the desired compound 5 were unsuccessful. Thus, aniline was protected with sodium formaldehydebisulfite,16,17 followed by coupling with diazonium salt 6 to yield compound 4. Alkaline hydrolysis of the protected amino derivative4with NaOH solution provided aniline derivative5 in 73% yield (Scheme 2). While 4-[4-(2-di- ethylamino-ethylamino)-1-naphthylazo]-benzenesulfon-amide18 as our reference inhibitor 1 was synthesized according to procedures in the literature19,20(Scheme 3). The proposed structural assignments were confirmed by detailed 1H, 13C NMR (HMQC, HMBC, COSY), and HR-EIMS analyses.
The Ab40 aggregation inhibitory activities of the syn-thetic compounds were measured using a thioflavin-T (ThT) fluorescence binding assay. As shown inFigure 3, the compounds were tested first at a high concentration (100 mM) and the relative ThT fluorescence, and hence, inhibition of Ab40 aggregation was determined. Results showed that compounds 1–5 significantly suppressed ThT fluorescence. In addition, the neurotoxicities were measured on cortical neurons using a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay.21 23After incubation of cortical neurons Figure 1.
Figure 2.
Figure 3. ThT fluorescence at Ex 450/Em 482 of Ab40aggregation. The compounds (100 mM) and Ab40(25 mM) were mixed in 50 mM TBS at pH 7.4 and incubated at 37C for 2 days, which promotes
aggregation of Ab40. The figure shows the meansS.D. (n=3). All data were scaled with the wild type (WT) response of Ab40 alone (100% fluorescence).
Scheme 1. Synthesis of phenylazo benzenesulfonamide derivatives 2 and3.
Scheme 2. Synthesis of b-amyloid inhibitors4and5.
Scheme 3. Synthesis of b-amyloid inhibitor1. 1174 S.-J. Lin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1173–1176
with MTT at 37C for 1 h, the medium was removed
and the formazan particles were dissolved with DMSO. OD600 nm was measured by ELISA reader. The ther-apeutic index of these compounds was determined as follows. (1) Ab40 aggregation inhibitory activity was determined using ThT binding assay at various con-centrations of compounds and the EC50 values were calculated. (2) Neurotoxicity was determined using the MTT reduction assay at various concentrations of compounds and the IC50values were calculated. (3) The therapeutic activity was determined as therapeutic index (TI, IC50/EC50). The reference compound1was included during each bioassay for comparison.11 Data are pre-sented inTable 1.
As shown in Table 1, we successfully identified novel small-molecule inhibitors for inhibition of Ab40 aggre-gates. The present structure–activity relationships (SAR) demonstrate that modification on the end of the phenyl-azo moiety at the para-position with different amino substituents results in significant effects on the Ab40 aggregation inhibitory activity and neurotoxicity. Results of these SAR conclusions are discussed below. (i) A simple phenylazo-like structure afforded good activity. (ii) None of the trans-stilbene benzenesulfon-amide derivatives displayed Ab40aggregation inhibitory activity (data not shown). (iii) Replacing the 4-(2-N,N-diethylamino-ethylamino)-naphthylamino moiety in 1 with a p-N,N-dimethylamino-phenylazo group in2 (LB-152) did not significantly decrease activity, as reflected in the relative EC50 values of 15.11 and 12.89 mM, respectively. However, the bulkier naphthylazo1showed increased neurotoxicity with an IC50of 22.14 3.01 mM as compared to IC50>100 mM with phenylazo derivatives2–
5. Consequently, LB-152 had a TI value > 7.75, which is approximately 5.3-fold higher than the TI value of 1.47 for compound 1. Additional mechanism studies are ongoing to better understand this result. (iv) Introdu-cing the tertiary amino group showed the strongest Ab40 aggregation inhibitory activity compared to the primary or secondary amino group (2versus3–5). These results suggested that the relatively high lipophilicity of the p-N,N-dimethylamino group (an electron-donating sub-stituent) on the end of the phenylazo moiety was very important for Ab40aggregation inhibitory activity. In summary, we have synthesized and evaluated a new series of para-substituted phenylazo benzenesulfon-amides as potential Ab40 aggregation inhibitors. The
p-N,N-dimethylamino substituted LB-152 possessed the highest Ab40 aggregation inhibitory activity without inducing neurotoxicity.
Inhibition of Ab40 aggregation and reduction in neuro-toxicity are expected to slow down or to arrest the pro-gress of AD, as well as to prevent the earliest form of Ab deposition. As mentioned earlier, one of the challenges in developing new inhibitors is to find nontoxic, non-peptide small-molecule lead compounds that can cross the BBB. Our studies have identified LB-152 as a useful lead compound for future design of promising clinical trials candidates for prevention and/or retardation of amylo-idogenesis involved in the development of AD. A more detailed analysis of the profile of LB-152 as a potent b-amyloid aggregation inhibitor for treatment of AD will be presented in due course.
Acknowledgements
We gratefully thank the National Science Council of Republic of China grants NSC-89-2314-B-077-013 (L.-M.Y.) and NSC-92-2320-B-038-040 (S.-J.L.) and the National Research Institute of Chinese Medicine for providing financial support.
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Compd R EC50(mM)b IC50(mM)c TI (IC50/EC50)d
1 4-NH(CH2)2NEt2 15.115.15 22.14 3.01 1.47
2 (LB-152) 4-NMe2 12.890.83 >100 >7.75
3 2-SMe, 4-NH2 56.797.79 >100 >1.76
4 4-NHCH2SO3Na 77.239.35 >100 >1.29
5 4-NH2 >100 >100 no suppression
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bEffect on fibril formation; concentration which inhibits wild type aggregation by 50%. cNeurotoxicity; concentration which inhibits viability of neurons by 50%.
dTherapeutic index, TI=IC 50/EC50.
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